1. Field of the Invention
Peroxidase is an enzyme which has a large number of desirable properties. It is available, inexpensive, stable to a substantial range of chemical and physical conditions and has a high turnover rate. Besides its obvious use for the determination of peroxidase substrates, it has found ample application in diagnostics for the localization of determinant sites in tissue and the determination in physiological media of a wide variety of analytes. Many analytes are present only in extremely small concentrations and high amplification of the signal is required as a result of a single binding event between the analyte and its homologous binding pair member. Frequently the number of enzyme molecules that signal a binding event is fixed based on the conditions of the assay and improved sensitivity or enhanced signal level is dependent upon a substrate which has a high turnover rate and provides a high signal level.
In developing substrates, there are many constraints. While peroxidase has relatively low specificity, there are limitations on the compounds which can be oxidized. Furthermore, while either the substrate or the product may in principle provide the signal, it is desirable to go from a low signal to a high signal. Therefore, the substrate should contribute little if any detectable signal where the product is measured. Other considerations include the solubility of the substrate and the interaction with materials which may be encountered in the samples to be measured. Additional considerations include ease of synthesis, stability (both shelf-life and in the assay medium), the effect ofthe production of side products (or even the desired product) on the activity of the peroxidase, and the ability to measure the product providing the detectable signal.
2. Brief Description of the Prior Art
Saunders and Watson, Biochem. J. (1950) 46:629-633, describe methoxy-substituted aniline as a peroxidase substrate. Keston and Brandt, Anal. Biochem. (1965) 11:1-5, describe fluorogenic substrates and products for use with peroxidase. Zaitsu and Ohkura, Ibid. (1980) 109:109-113 describe a variety of phenolic compounds for use in a fluorimetric assay with peroxidase. A wide variety of phenolic compounds and amines have been reported as chromogenic and fluorogenic substrates for peroxidase.